We propose a novel and exciting hypothesis?based on literature and our own pilot data?that neuronal injury stimulates astrocyte activation via thrombin and its receptor PAR-1, and the resulting astrocyte response to injury yields transmissible paracrine protection of neurons. Together, neurons, astroctyes, endothelial cells, pericytes, and oligodendroglia comprise the neurovascular unit, or NVU. Data generated from the several aims will yield insights into the mechanism for the well-documented phenomenon of pre-conditioning; will provide considerable insights into the mechanisms of differential vulnerability to cerebral injury; and would likely yield treatment options for stroke patients. To accomplish the aims, we are creating novel conditional knock-out mice that will provide research tools to the broader research field. The previous award, R01 NS075930, covered 8/15/2011 to 03/31/2015 and involved 4 aims. All proposed work has been completed and published or under review. The data from our prior award has already influenced stroke care significantly, in the form of two funded clinical trials. However, our prior data could not fully explain the known differences among the elements of the NVU, specifically, that astrocytes and endothelial cells tolerate ischemia better than neurons. What is the mechanism of thrombin-mediated pre-conditioning, and can we relate that mechanism to other forms of pre- and peri-conditioning? Why does the brain contain prothrombin mRNA and protein and what purpose could be served by thrombin activation in brain? Could we derive insights about the response of the brain to injury, and specifically explore the cell-subtype interactions during injury among astrocytes, neurons, pericytes, oligodendroglia, and endothelial cells? To answer these questions, and to address the central hypothesis of the present award application, we added significant new models to our repertoire, and generated pilot data speaking to the feasibility and novelty of the proposed new specific aims.